Tuberous sclerosis (TSC) is an autosomal dominant tumor suppressor gene syndrome, characterized by development of distinctive benign tumors (hamartomas) and malformations (hamartias) in multiple organ systems, due to mutations in either TSC1 or TSC2. The long-term objectives of this project are to understand in detail the functions of the TSC1 and TSC2 genes and proteins, their interacting partners, how they are regulated, and how their loss leads to deregulated growth control in the tumors that develop in TSC patients. The insight achieved will have relevance to growth regulatory mechanisms in general, including in malignant cells. There are four specific aims in this project. First, the importance of phosphorylation at multiple sites on TSC2 by the Akt kinase will be critically tested in vivo through generation and analysis of an allele in the mouse in which these sites are mutated to Ala. Second, we will examine the mechanism and importance in growth control of feedback circuitry in the TSC1/TSC2 signaling pathway that serves to attenuate activation of the Akt kinase when TSC1/TSC2 are lacking, in both cell lines and tumors. Third, we will explore the estrogen responsive growth that is seen in TSC1/TSC2 null cell lines, examining its mechanism, effects on ERa expression and phosphorylation, consequences in terms of gene transcription, and in vivo effects in tumor growth in mice. Fourth, we will examine whether Rheb is the sole downstream effector that is perturbed when TSC1/TSC2 are lacking from cells, whether Rheb activation has the identical effects in cells as the loss of TSC1/TSC2, and identify binding partners of Rheb for downstream signaling effects.